Abstract:

Objective To discuss the improved methods for the isolation and culture of primary chondrocytes from the neonatal rats，and to establish an efficient and economical in vitro chondrocyte culture system. Methods The primary chondrocytes were isolated from the joints of neonatal rats and divided into overnight digestion （OD） group and rapid digestion （RD） group for separation. The chondrocytes in OD group were digested overnight by typeⅡ collagenase， while the chondrocytes in RD group were separated by the combination of pre-digestion with physical and chemical digestion methods. The chondrocytes were cultured in modified media containing 0% （blank group 1）， 1%， 2%， 4%， and 10% fetal bovine serum （FBS）， 0 （blank group 2）， 0.1， 0.2， 0.4， 0.8， 1.0， and 2.0 g·L^-1 vitamin C（VC）， and 0 （blank group 3）， 0.5， 1.0， 2.0， 4.0， 8.0， 10.0 μg·L^-1 poly（lactic-co-glycolic acid） （PLGA） nanoparticles. The media containing different concentrations of FBS， VC， and PLGA were mixed with Dulbecco’s modified Eagle’s medium/nutrient mixture F-12（DMEM/F12）， and were divided into related groups based on the concentrations of ingredients. Cell counter was used to count the chondrocytes in various groups and the survival rates and diameters of the chondrocytes in various groups were detected； Toluidine blue staining was used to detect the morphology of the chondrocytes in various groups； CCK-8 method was used to detect the proliferative activities of the chondrocytes in various groups； cell adhesion assay was used to detect the adhesion rates of the chondrocytes in various groups； Hoechst/propidium iodide（PI） staining was used to detect the apoptosis of the chondrocytes in various groups； MTT assay was used to detect the proliferation activities of the chondrocytes in various groups after treated with modified media.The cells were divided into DMEM/F12+10%FBS group， DMEM/F12+1%FBS group， and DMEM/F12+1% FBS+0.4 g·L^-1 VC+1 μg·L^-1 PLGA group. Real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of sex-determining region Y-box 9 （SOX9）， collagen type Ⅱ alpha 1 chain （Col2A1）， collagen type Ⅹ alpha 1 chain （Col10A1）， and matrix metallopeptidase 13 （MMP13） mRNAs in the chondrocytes in various groups after treated with modified media；immunofluorescence staining was used to detect the expressions of type Ⅱ collagen （COLⅡ） and SOX9 in the chondrocytes in various groups after treated with modified media. Results The survival rate of primary chondrocytes in OD group was lower than that in RD group， and the average cell diameter was larger than that in RD group. The primary chondrocytes in OD group were larger and spindle-shaped， and most cells exhibited pseudopodia； in RD group， the primary chondrocytes were smaller， mostly rhomboid in shape， with only a portion of the cells showing pseudopodia. The Toluidine blue staining results showed significant coloration in both groups， but the digestion time of the chondrocytes in RD group was shorter， and compared with OD group， the actual culture time of the chondrocytes was reduced by 9-13 h， and more immature morphology of the primary chondrocytes were observed. The proliferation activity of the primary chondrocytes in OD group was slow at 24 h of culture but increased at 48 h of culture， and the proliferation activity of the primary chondrocytes was significantly higher at 48 h of culture compared with 12 h of culture （P<0.01）. Compared with 12 h of culture，the proliferation rates of the primary chondrocytes in RD group were increased at 24 and 48 h of culture （P<0.01）. At 24 and 48 h of culture， compared with OD group， the proliferation rates of the primary chondrocytes in RD group were increased （P<0.05）. The number of apoptotic chondrocytes in RD group was lower than that in OD group， and no necrotic chondrocytes were observed in either group. The proliferation activities of chondrocytes of the rats were increased with the rising of FBS concentration in the culture medium. Compared with blank group 1， the proliferation activities of chondrocytes of the rats after treated with culture mediums containing 1%， 2%， 4%， and 10% FBS were significantly increased （P<0.05）. Compared with blank group 2， the proliferative activities of chondrocytes of the rats after treated with culture mediums containing 0.2-1.0 g·L^-1 VC were significantly increased （P<0.05）， and the highest proliferation activity was found when the concentration of VC was 0.4 g·L^-1 （P<0.01）. Compared with blank group 3， the proliferation activities of chondrocytes of the rats after treated with culture mediums containing 1-4 μg·L^-1 PLGA were significantly increased （P<0.05）， and the highest proliferation activity was found after treated with culture medium containing 1 μg·L^-1 PLGA （P<0.05）. Compared with DMEM/F12+10%FBS group， the expression levels of SOX9 mRNA and Col2A1 mRNA in the chondrocytes in DMEM/F12+1%FBS group were significantly increased （P<0.05 or P<0.01）. Compared with DMEM/F12+10%FBS group， the expression levels of SOX9 mRNA and Col2A1 mRNA in the chondrocytes in DMEM/F12+1%FBS+0.4 g·L^-1 VC+1 μg·L^-1 PLGA group were significantly increased （P<0.01）. The immunofluorescence staining results showed that the green fluorescence signal of COLⅡ and the red fluorescence signal of SOX9 were observed in some chondrocytes in DMEM/F12+10%FBS group under fluorescence microscope， and the fluorescence intensity was weak. In DMEM/F12+1%FBS group， most chondrocytes exhibited COLⅡ green fluorescence signal and SOX9 red fluorescence signal， and the fluorescence intensity was significantly stronger than that in DMEM/F12+10% FBS group. In DMEM/F12+1% FBS+0.4 g·L^-1 VC+1 μg·L^-1 PLGA group， the COLⅡ green fluorescence signal and SOX9 red fluorescence signal were found in all the chondrocytes， and the fluorescence intensity was significantly higher than those in DMEM/F12+10%FBS and DMEM/F12+1%FBS groups. The expression levels of COLⅡ and SOX9 proteins in the chondrocytes in DMEM/F12+1%FBS group were significantly higher than those in DMEM/F12+10%FBS group， and the expression levels of COLⅡ and SOX9 proteins in the chondrocytes in DMEM/F12+1%FBS+0.4 g·L^-1 VC+ 1 μg·L^-1 PLGA group were significantly higher than those in DMEM/F12+10%FBS group. Conclusion The improved methods for the isolation and culture of primary chondrocytes of the rats can overcome the shortcomings of traditional methods， shorten the isolation time of primary chondrocytes， and improve the quality of in vitro culture of primary chondrocytes.

Key words: Chondrocytes, Primary cell culture, In vitro techniques, Conditioned culture media, Serum